During the past grant cycle, our gene therapy studies showed that genes encoding enzymes that detoxify reactive oxygen species (ROS) have a protective effect on suppressing experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Antioxidant gene therapy restored integrity to the blood-brain barrier (BBB) and reduced demyelination of the optic nerve. However, axonal and neuronal degeneration rather than demyelination are now increasingly recognized as the primary causes of visual and neurological disability. Currently, there is no effective treatment to suppress or repair this damage. Moreover, the mechanisms leading to neurodegeneration in optic neuritis and MS are poorly understood. We have found that peroxynitrite mediates nitration of key mitochondrial proteins. This commences during the earliest phase of the EAE animal model of MS, well before infiltration of inflammatory cells. Mitochondrial oxidative injury in EAE animals resulted in apoptosis of ganglion cells of the retina and oligodendrocytes in the optic nerve. These novel findings implicate mitochondrial ROS activity as a key event in the initiation of optic nerve degeneration and demyelination in EAE. Mitochondria play a key role in the pathogenesis of many neurological diseases, but the role of the organelle has only recently been recognized in MS. Here we propose a hypothesis that nitration of key mitochondrial proteins leads to the mitochondrial dysfunction which plays a pivotal role in the neurodegeneration of optic neuritis and MS. Our first two specific aims will explore the role of mitochondrial bioenergetics and oxidative stress, focusing on the import and function of mitochondrial proteins in the optic nerves of mice induced with EAE and transgenic TCR MOG mice that spontaneously develop optic neuritis. In Aim III we develop a strategy to repair the damage by testing the effects of modulating mitochondrial gene expression by complementation with AAV mediated delivery of the defective subunits to the retina and optic nerve. We will use the new technolology to treat patients with optic neuritis and multiple sclerosis in a future grant cycle. PUBLIC HEALTH RELEVANCE: There is no effective treatment for the optic nerve degeneration and persistent visual loss that occurs in people who have optic neuritis. Nor is there an effective treatment to prevent recurrences of optic neuritis and additional loss of vision. Optic neuritis is also often the first sign of multiple sclerosis that is the leading cause of disability in young adults in the United States. The proposed study in mice is designed to develop a strategy to treat patients at risk for axonal degeneration and persistent visual loss from optic neuritis and multiple sclerosis.